SUMMARY/ABSTRACT Mass spectrometry (MS) has played a leading role in the past three decades in the field of proteomics. A combination of innovative MS-based techniques has provided powerful tools for proteomic discovery including the ability to identify protein biomarkers in a complex sample, quantify changes in protein expression under different conditions including those related to disease, and characterize protein-protein interactions which play a key role in complex cellular pathways. A second important advance in MS has been the introduction of mass spectrometric imaging (MSI) which extends MS to the spatial dimension, allowing mapping of the distribution of biomolecules including proteins, nucleic acids, metabolites and even small drug compounds in complex tissues. The goal of this Phase I project is to enable MSI to perform highly multiplexed, nanoscale imaging of targeted biomolecules in biospecimens. Such a capability would provide a major tool for systems biology, which requires detailed knowledge of the structure and structural changes of complex tissues at the molecular, subcellular and cellular levels. It would also provide critical new information for cancer research and for therapeutics, diagnostics and monitoring of cancer patients where targeted biomolecules in fresh frozen (FF) or formalin fixed paraffin embedded (FFPE) thin sections of cancer tissue are imaged. However, several difficult challenges remain before routine, highly multiplexed nanoscale MALDI-MSI of biospecimens is possible including: i) developing highly sensitive and selective MSI- compatible multiplex mass-tag probes for hundreds of biomolecules including proteins and miRNAs and ii) developing methods to obtain nanoscale resolution with MSI. In order to solve these problems, we will explore the use of our newly developed class of improved photocleavable mass-tags (iPC-MTs) during Phase I. Initial studies show that iPC-MTs have superior properties such as higher sensitivity compared to earlier developed mass-tags and can facilitate simultaneous identification of hundreds of targeted biomolecules in standard FF and FFPE tissue slices. In contrast, conventional immunofluorescent antibodies and fluorescently labeled DNA/RNA hybridization probes used with light microscopy can only detect a few target biomolecules simultaneously. In addition, iPC-MTs can incorporate ?polymer tethering? groups making them compatible with the newly developed method of expansion microscopy (ExM) which offers the potential to obtain nanometer spatial resolution using MSI. This approach involves the physical isotropic swelling of biospecimens such as FFPE thin sections by embedding into them hydrogel polymers. The application of iPC-MTs can thus enable multiplexed nanoscale MSI imaging. This work will be facilitated by our collaboration with leading experts in the MALDI-MSI and ExM fields including Drs. Cathy Costello (BU, William Fairfield Warren Distinguished Professor, Director of BU Center for Biomedical Mass Spectrometry), Ed Boyden (MIT, Y. Eva Tan Professor of Neurotechnology) and Jason Amsden (Duke University, Assistant Research Professor, Nanomaterials and Thin Films Lab at the Pratt School of Engineering).